ADP-ribosylation factors (ARFs) are a family of about 20-Kda guanine nucleotide- binding proteins that were originally identified by their ability to stimulate in vitro cholera toxin-catalyzed ADP-ribosylation of Gsalpha, the alpha subunit of the stimulatory heterotrimeric GTP-binding protein of the adenylyl cyclase system. More recently, ARFs have been implicated in intracellular protein trafficking and are thought to be involved in the assembly of non-clathrin coated vesicles. Based on molecular cloning, at least six mammalian ARFs are known. These fall into three classes based on deduced amino acid sequence, size, phylogenetic analysis, and gene structure. The ARFs from the three classes differ in tissue-specific expression and developmental regulation. The 5'-flanking region of the human ADP-ribosylation factor (ARF) 3 gene contains the features of a housekeeping gene. It lacks a TATA or CAAT box, has several GC-boxes within a highly GC-rich region, and utilizes multiple transcription initiation sites. The cis-acting elements involved in regulating expression of the gene were identified by transient transfections of IMR-32 neuroblastoma cells. Reporter plasmids were modified to facilitate construction of defined promoter deletions linked to chloramphenicol acetyltransferase or luciferase using ligation-independent cloning. Transfection analyses indicated that sequences within 58 bp of the transcription initiation site were necessary for full expression, in particular, a sequence containing the 10-bp palindrome TCTCGCGAGA. Electrophoretic mobility-shift assays performed with IMR-32 nuclear extracts demonstrated that a DNA-binding protein, termed TLTF, bound to an oligonucleotide containing this palindrome. Competition experiments showed that mutations within the core of the palindrome abolished in vitro binding and that the same protein bound to a 5~-proximal sequence. Expression of the promoter containing a mutated palindrome was reduced dramatically, consistent with the conclusion that this region functions in vivo to control expression of the ARF3 gene.